| At present, the sources of taxol, which is successful antineoplastic drug widely used in the effective treatment of ovarian and breast cancers, are very tense. In order to secure a long-term supply of taxol, large-scale taxus cell culture provides a promising process, but having not yet been used for the production of taxol. The main problem is the unstable production of taxol during the cell culture, which makes the cost of large-scale cell culture to produce taxol is still higher than the natural extraction's, so as to prevent its industrial process. Specific synthesis of secondary metabolites or not and the yield of its synthesis are decided mainly by a series of key enzymes'expression and activity during its metabolic synthesis pathway. Transcriptional regulation is the main mode of eukaryotic gene regulation. Through renovating promoter of the key enzyme genes or transferring corresponding transcription factors, gene expression of plant secondary metabolites synthase can be activated, which can effectively improve the yield of target secondary metabolites. In this paper, a promoter of taxol biosynthetic key-eneyme gene DBAT of taxus media is cloned, and its function is studied, which lay the base for cloning corresponding transcription factors.The main results are as following:1 A method of inducing callus of Taxus media effectively is established.When the media is MS+0.3 mg/L 2,4-D+0.4 mg/L NAA+30 g/L sucrose+8 g/L agar,pH 5.8-6.0, the stems of branches of one-year old collected in August as explants have the better inducement than in May and in October; the stems of one-year old branches collected in August as explants are better than those of two years old branches; the stems of branches of one-year old collected in August as explants dealt with a litter petiole have the highest inducement;2 Establishing a genomic DNA extraction method for taxus media cells. Through three methods of plant genomic DNA extraction, genomic DNA of taxus media cells is extracted. From the experiment, an effective method which can extract high quality and molecular weight DNA from taxus media cells is found: DNA extraction method of SDS-CTAB can gain taxus media cell genomic DNA with better concentration and purity than SDS and CTAB, which lays foundation for the further molecular biological researchs;3 Establishing the suitable PCR system for amplification of genomic DNA of taxus media cells:15μl system——Template 20 ng/μl; dNTP(10 mM)0.3μl; MgCl2 (25 mM)0.8μl; Taq DNA polymerase (2.5 U/μl)0.3μl; 10×buffer 1.5μl; Primer(10μmol/μl)0.2μl; ddH2O 11.6μl. Procedures: 95℃4 min(1 cycle); 93℃1 min, 39℃1 min40 s, 72℃2 min20 s (35 cycles); 72℃10 min (1 cycle); 4℃10 min (1 cycle);4 A new method of cloning promoters based on RAPD is established, and a promoter sequence of taxol biosynthetic key enzyme gene DBAT is cloned with the moehod successfully, which is 1054 bp length. Analyzed with softwares, the sequence has the own structure of promoters.To analyse the function of this promoter,a plant expression vector with GUS reporter gene were constructed by fusing this prometer with pCAMBIA1381,which is based for analyzing its function by transforming into tobacco cells next.
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